Gene expression and proteomic analysis of the formation of Phakopsora pachyrhizi appressoria

Abstract

BackgroundPhakopsora pachyrhizi is an obligate fungal pathogen causing Asian soybean rust (ASR). A dual approach was taken to examine the molecular and biochemical processes occurring during the development of appressoria, specialized infection structures by which P. pachyrhizi invades a host plant. Suppression subtractive hybridization (SSH) was utilized to generate a cDNA library enriched for transcripts expressed during appressoria formation. Two-dimensional gel electrophoresis and mass spectroscopy analysis were used to generate a partial proteome of proteins present during appressoria formation.ResultsSequence analysis of 1133 expressed sequence tags (ESTs) revealed 238 non-redundant ESTs, of which 53% had putative identities assigned. Twenty-nine of the non-redundant ESTs were found to be specific to the appressoria-enriched cDNA library, and did not occur in a previously constructed germinated urediniospore cDNA library. Analysis of proteins against a custom database of the appressoria-enriched ESTs plus Basidiomycota EST sequences available from NCBI revealed 256 proteins. Fifty-nine of these proteins were not previously identified in a partial proteome of P. pachyrhizi germinated urediniospores. Genes and proteins identified fell into functional categories of metabolism, cell cycle and DNA processing, protein fate, cellular transport, cellular communication and signal transduction, and cell rescue. However, 38% of ESTs and 24% of proteins matched only to hypothetical proteins of unknown function, or showed no similarity to sequences in the current NCBI database. Three novel Phakopsora genes were identified from the cDNA library along with six potentially rust-specific genes. Protein analysis revealed eight proteins of unknown function, which possessed classic secretion signals. Two of the extracellular proteins are reported as potential effector proteins.ConclusionsSeveral genes and proteins were identified that are expressed in P. pachyrhizi during appressoria formation. Understanding the role that these genes and proteins play in the molecular and biochemical processes in the infection process may provide insight for developing targeted control measures and novel methods of disease management.

Overview of the functional virulent genome of the coffee leaf rust pathogen Hemileia vastatrix with an emphasis on early stages of infection

“…In the same way, the comparison of the 9234 H. vastatrix transcript to a collection of ESTs and proteins differentially expressed in P. pachyrhizi appressoria (Stone et al, 2012) shows a very limited number of genes in common between both studies (Supplementary Data 14). Interestingly however, the comparison of 4492 P. pachyrhizi haustorial ESTs (Link et al, 2013) to the 9234 H. vastatrix transcripts identified 1668 hits to 1132 unique H. vastatrix transcripts.…”

New insights into Phakopsora pachyrhizi infection based on transcriptome analysis in planta

“…We conducted an enrichment analysis in Blast2GO to identify GO terms that were overrepresented in each genotype for which P. pachyrhizi reads were obtained, susceptible (BRS231) and resistant (PI561356), as well as GO terms that were conserved and overrepresented among different infection structures, such as germinated urediniospores and appressoria ( Stone et al , 2012 ), haustoria ( Link et al , 2014 ), and leaf lesions.…”

“…Since the nuclear genome of P. pachyrhizi has not yet been sequenced, most of the molecular information about this pathogen has been obtained from work involving transcriptome sequencing associated with bioinformatics analyses. Thus, a moderate repertoire of candidate gene sequences expressed in germinated urediniospores, appressorium, haustorium, and uredinia have become available ( Posada-Buitrago and Frederick, 2005 ; Tremblay et al , 2009 , 2012 , 2013 ; Stone et al , 2012 ; Link et al , 2014 ; Carvalho et al , 2016 ). Most of these studies focused on the characterization of specific molecular mechanisms during the infection process, with a concentration on analyses of specific structures of the pathogen, such as the urediniospores and haustorium, or of the total infected leaf.…”

Advances in understanding obligate biotrophy in rust fungi

“…Dikaryotic aeciospores and urediniospores enter the host leaf through openings in the stomata, after appressorium differentiation (Voegele et al, 2009). Several transcriptomic analyses of appressoria formation of urediniospores led to identification of the genes responsible for metabolism and regulation of the cell division cycle, indicating that this structure plays an active role in the penetration process (Hu et al, 2007;Stone et al, 2012;Talhinhas et al, 2014). Urediniospores from the soybean rust fungus directly penetrate their host through the epidermis, in a similar manner to basidiospores of heteroecious fungi, and form an appressorium with a high cell turgor pressure of 5.13 MPa (Loehrer et al, 2014a), which correlates with what has been observed in other fungal plant pathogens (Ryder & Talbot, 2015).…”